The present invention relates to an aldehyde oxidase gene derived from a plant and utilization thereof.
It has been known that a natural plant growth hormone auxin alternatively IAA or indoleacetic acid is produced from tryptophane via indoleacetaldehyde followed by the action of anoxidase in higher plants. The hormone is deeply involved in various morphogenesis and environmental adaptation of a plant by its physiological activity and has significant effects on maturing by growth acceleration in general crop cultivation, improvement in yield and in quality by rooting acceleration in nursery plant production, increase in yield by growth acceleration of fruits in fruit vegetable cultivation, increase in added value by acceleration of flowering and elongation of life by prevention of defoliation or aging in ornamental plant cultivation. Therefore, there has been a strong demand for a method for artificially controlling the said enzyme for industry and particularly agricultural production.
Under these circumstances, the present inventors have successfully determined the total amino acid sequence and gene of the enzyme and completed the present invention.
Thus, the present invention provides:
1) An aldehyde oxidase gene which is a 4.4 Kbp gene obtainable from a plant and which encodes an amino acid sequence of an enzyme capable of oxidizing an aldehyde compound to a carboxylic acid (hereinafter, referred to as the gene of the present invention),
2) The aldehyde oxidase gene according to item 1), wherein the aldehyde compound is indoleacetaldehyde and the carboxylic acid is indoleacetic acid,
3) The aldehyde oxidase gene according to item 1 or 2 which is derived from a maize plant (Zea mays L.),
4) The aldehyde oxidase gene according to item 1 which is a nucleotide sequence encoding an amino acid sequence shown by SEQ ID NO: 2,
5) The aldehyde oxidase gene according to item 4 which has a nucleotide sequence shown by SEQ ID NO: 1 (loci of CDS being 46 . . . 4120),
6) The aldehyde oxidase gene according to item 1 which is a nucleotide sequence encoding an amino acid sequence shown by SEQ ID NO: 4,
7) The aldehyde oxidase gene according to item 6 which has a nucleotide sequence shown by SEQ ID NO: 3 (loci of CDS being 91 . . . 4138),
8) A plasmid comprising the aldehyde oxidase gene according to item 1, 2, 3, 4, 5, 6 or 7,
9) A transformant transformed by introducing the plasmid according to item 8 into a host cell,
10) The transformant according to item 9, wherein the host cell is a microorganism,
11) The transformant according to item 9, wherein the host cell is a plant,
12) A process for constructing an expression plasmid which comprises ligating:
(1) a promoter capable of functioning in a plant cell,
(2) an aldehyde oxidase gene according to item 1, 2, 3, 4, 5, 6 or 7 and
(3) a terminator capable of functioning in a plant in a functional manner and in the said order described above,
13) An expression plasmid comprising:
(1) a promoter capable of functioning in a plant cell,
(2) an aldehyde oxidase gene according to item 1, 2, 3, 4, 5, 6 or 7 and
(3) a terminator capable of functioning in a plant which are ligated in a functional manner and in the said order described above,
14) A process for controlling production of an aldehyde oxidase in a transformant which comprises introducing, into a host cell, an expression plasmid comprising:
(1) a promoter capable of functioning in a plant cell,
(2) an aldehyde oxidase gene and
(3) a terminator capable of functioning in a plant which are ligated in a functional manner and in the said order described above to transform said host cell,
15) The process according to item 14, wherein the aldehyde oxidase gene is derived from a plant and the host cell is a plant, and
16) The process according to item 14, wherein the expression plasmid is the expression plasmid according to item 13.
The present invention will be described in more detail.
The gene of the present invention comprises about 4.4 kbp nucleotide obtainable from a plant and is an aldehyde oxidase gene that encodes an amino acid sequence of an enzyme capable of oxidizing an aldehyde compound to generate a carboxylic acid. For example, it is capable of oxidizing indoleacetaldehyde to generate indoleacetic acid.
The gene of the present invention can be obtained from a plant, for example, maize or the like. The gene of the present invention and the enzyme as the translation product of it have an action of oxidizing an acetaldehyde compound to a carboxylic acid in a cell. Said enzyme may also act, for example, on benzaldehyde, butyraldehyde, protocatechualdehyde or the like as the substrate, in addition to indolealdehyde of course, a single enzyme may act on plural compounds as substrates.
The gene of the present invention specifically includes, for example, a gene which is a nucleotide sequence encoding an amino acid sequence shown by SEQ ID NO: 2 and a gene which is a nucleotide equence encoding an amino acid sequence shown by SEQ ID NO: 4 as well as an equivalent of them. The expression xe2x80x9can equivalent of themxe2x80x9d used herein means an aldehyde oxidase gene having a nucleotide sequence of an aldehyde oxidase gene that encodes an amino acid sequence shown by SEQ ID NO: 2 kor SEQ ID NO: 4 with a single nucleotide or plural nucleotides added, deleted or replaced, and refers to a DNA which is an analog having the same function. More particularly, this includes a gene having a nucleotide sequence shown by SEQ ID NO: 1 (loci of CDS being 46 . . . 4120) or a nucleotide sequence shown by SEQ ID NO: 3 (loci of CDS being 91 . . . 4138).
The gene of the present invention can be obtained by the following process.
For example, seeds of Golden Cross Bantam 70 (purchased from Sakata-no-tane), a maize cultivar, are subjected to a treatment for hastening of germination by immersing overnight in running tap water, subsequently seeded on a paper towel moistened with water and placed in red light (0.8 W/m2) under a condition of 25xc2x0 C. for 2 days and then in the dark for 1 day to allow germination. Top portions of young sheaths grown to 1.0-1.5 cm from the obtained seedlings are excised under a green safety light, immediately frozen with liquid nitrogen and stored at xe2x88x9230xc2x0 C. as samples for purification of enzymes and samples for extracting RNAs.
For purifying aldehyde oxidase from the frozen samples prepared in this manner, it is appropriate to use a method described in T. Koshiba et al., Plant Physiology, 1996, 110, 781-789.
In order to prevent decrease in activity of the enzyme and decomposition of the protein during procedures of extraction and purification, it is preferred to carry out all the treatments in the purification steps at a lower temperature of 2-4xc2x0 C., as is ordinary manner in such procedures. First, 150-200 g of the frozen sample is taken as a material for one batch of purification. The material is mechanically crushed by a homogenizer or the like with addition of 400 ml of 0.1 M phosphate buffer (pH 7.4) and centrifuged at 12,000 g for 30 minutes. The supernatant is separated as a crude enzyme standard sample. From the crude enzyme standard sample, a fraction is obtained with 30-50% saturated ammonium sulfate, dialyzed against 20 mM Tris HCl buffer (pH 8.0) and centrifuged at 20,000 g for 20 minutes. The supernatant from centrifugation is passed over an ion-exchange column (for example, DEAE TOYOPEARL 650 M, manufactured by Tosoh) and a fraction with an aldehyde oxidase activity is collected. Said fraction with the specific activity is subjected to chromatography with a hydrophobic column, a hydroxyapatite column and an ion-exchange column (for example, DEAE-5PM) in this order and purified until the fraction with aldehyde oxidase activity is detected as an almost single protein band by silver staining after electrophoresis.
According to the above described purification procedure, about 2,000 times purification, in terms of the amount of protein in the crude enzyme standard sample, is usually possible. It can be confirmed that the finally purified protein has a size of about 300 kD in molecular weight by the gel filtration column process. Further, it can be detected as a band having a size of about 150 kD in molecular weight by SDS polyacrylamide gel electrophoresis (SDS-PAGE), indicating that said enzyme forms a dimer.
In the above described fractionating process by column chromatography, effective collection of the fraction with aldehyde oxidase activity can be achieved making use of measurement of aldehyde oxidase activity in respective fractions. For this purpose, a method in which indoleacetaldehyde is added to the purified fraction as a substrate and the amount of produced indoleacetic acid is determined by HPLC, for example, can be utilized. Precisely, 100 xcexcl of reaction solution consisting of 5-50 xcexcl of the purified fraction, 0.1 mM indoleacetaldehyde and 0.1 mM phosphate buffer (pH 7.4) is prepared. The solution is incubated at 30xc2x0 C. for 30 minutes to effect the reaction and, immediately after, 8 xcexcl of 1 N HCl, 5 xcexcl of 2.0 M sodium hydrogen sulfite and 50 xcexcl of methanol are added to the solution to quench the reaction. The reaction solution is centrifuged at 15,000 g for 5 minutes and 100 xcexcl of the obtained supernatant is taken as a analytical sample for HPLC. By detecting absorption at 280 nm, indoleacetaldehyde as the substrate and indoleacetic acid as the reaction product can be quantitatively analyzed. It is effective to carry out HPLC with, for example, ODS C18 column and to elute with 20-50% linear gradient of methanol containing 0.1% acetic acid.
The protein obtained in this manner is partially digested and the digested peptide is analyzed to obtain a partial amino acid sequence information. Usually, the purified aldehyde oxidase sample is separated by SDS-PAGE and a protein band of 150 kD is collected by excision. The collected gel fragments are treated, for example, with Achromobacter Protease I (API) in the presence of 0.1% SDS and digested peptide fragments are extracted. This is loaded, for example, on a reverse phase HPLC accompanied by a pre-column of an anion exchanger (DEAE) to separate peptides and recover them. The amino acid sequences are determined by a protein sequencer and parts of the samples are subjected to molecular weight determination by MALDI-TOF to check accuracy of the obtained amino acid sequence information.
Then, an oligo DNA expected to encode the amino acid sequence is synthesized on the basis of the obtained amino acid sequence information. Further, RT-PCR is conducted using a total RNA as a template to amplify cDNA partial fragment, which is then cloned into a plasmid vector.
For extraction of the total RNA, 7 g of the frozen sample, for example, is triturated in liquid nitrogen with a mortar and a pestle to form fine powders. After evaporating liquid nitrogen, RNA is extracted by the conventional manner, for example, using guanidine thiocyanate/cesium chloride process and the total RNA is collected from the extract by ethanol precipitation. By this procedure, usually 1 mg of the total RNA is obtained.
For amplification of cDNA, a reverse transcription reaction is carried out using, among synthetic oligo DNAs, one synthesized in antisense orientation as a primer and binding it to a transcription product of a target RNA contained in the total RNA. The reverse transcription reaction can be conducted using a commercially available reverse transcription PCR kit, for example, RNA-PCR kit (manufacturedbyPerkin-ElmerCetusInstruments). Then,the obtained reverse transcription product can be subjected again to PCR in which an oligo DNA synthesized in sense orientation is added to amplify cDNA fragment.
The obtained cDNA amplification fragment is purified and cloned into a plasmid vector. As the plasmid vector, for example, pCRII (manufactured by Invitrogen) can be used and cDNA amplification fragment can be cloned by transforming E. coli according to the conventional manner and screening transformants having an insert. The nucleotide sequence of the clone is determined using, for example, ABI PRISM Dye Primer Cycle Sequencing Ready Reaction Kits (manufactured by Applied Biosystems) on the obtained cDNA clone.
Sense and antisense primers for part of nucleotide sequence in cDNA partial fragment obtained in this manner can be synthesized and subjected to RACE to obtain cDNA fragments having terminals in 5xe2x80x2-orientation and 3xe2x80x2-orientation, respectively. A complete length cDNA can be obtained by ligating them and cloning into a plasmid vector. For the RACE, a commercially available Marathon cDNA Amplification Kit (manufactured by Clontech), for example, can be used.
The gene of the present invention can be utilized in the following manner.
For example, a host cell such as a microorganism, a plant or the like is transformed by introducing the gene of the present invention to form a transformant.
In order to introduce and express the gene of the present invention in a plant cell, an expression plasmid comprising (1) a promoter capable of functioning in a plant cell, (2) a gene of the present invention (an aldehyde oxidase gene described in items 1 to 7 above) and (3) a terminator capable of functioning in a plant cell which are ligated in a functional manner in a plant cell and in the said order described above and introduced in a plant cell to transform said cell.
The expression xe2x80x9cin a functional mannerxe2x80x9d used herein means that, when the constructed plasmid is introduced into a plant cell to transform it, the gene of the present invention is integrated under the control of a promoter such that the gene is normally transcribed/translated and have a function of expressing a protein in said plant cell.
The promoter capable of functioning in a plant cell includes, for example, T-DNA derived constitutive type promoters such as nopaline synthase gene (NOS) promoter, octopine synthase gene (OCS) promoter and the like, plant virus derived promoters such as cauliflower mosaic virus (CaMV) derived 18S and 35S promoters and the like, and inducible type promoters such as phenylalanine ammonialyase (PAL) gene promoter, chalcone synthase (CHS) gene promoter, pathogen-related (PR) gene promoter and the like. Further, it includes other known plant promoters.
The terminator capable of functioning in a plant cell includes, for example, T-DNA derived constitutive type terminators such as nopaline synthase gene (NOS) terminator and the like, plant virus derived terminators such as garlic virus GV1, GV2 terminators and the like. Further, it includes other known plant terminators.
For transforming a plant cell by introducing such plasmid into a plant cell, the above described expression plasmid is introduced into a plant cell by any of conventional means such as Agrobacterium infection method (JP-B-2-58917 and JP-A-60-70080), electroporation method into protoplast (JP-A-60-251887 and JP-A-5-68575), particle gun method (JP-A-508316 and JP-A-63-258525) and the like, and a transformed plant cell can be obtained by selecting a plant cell into which the gene of the present invention is introduced. The transformed plant is obtained by regenerating a plant according to a conventional plant cell culturing process, for example, described in Uchimiya, Manual for Plant Gene Manipulation (Method for Producing Transgenic Plants), Published by Kodansha Scientific (ISBN 4-06-153515-7 C3045), 1990, pages 27-55.
Further, the present invention provides a process for controlling production of an aldehyde oxidase in a transformant which comprises introducing, into a host cell, an expression plasmid comprising (1) a promoter capable of functioning in a plant cell, (2) an aldehyde oxidase gene and (3) a terminator capable of functioning in a plant which are ligated in a functional manner and in the said order described above to transform said host cell.
The promoter capable of functioning in a plant cell includes, for example, lacZ gene promoter of lactose operon in E. coli, alcohol dehydrogenase gene (ADH) promoter in yeast, Adenovirus major late (Ad.ML) promoter, early promoter of SV 40, Baculovirus promoter and the like. When the host is a plant, promoters capable of functioning in a plant as described above may also be included.
The terminator capable of functioning in a plant cell includes, for example, HIS terminator sequence in yeast, ADHI terminator, early splicing region of SV 40 and the like. When the host is a plant, terminators capable of functioning in a plant as described above may also be included.
The aldehyde oxidase gene may be any one insofar as it is a gene encoding an amino acid sequence of an enzyme capabre of oxidizing an aldehyde compound to form a carboxylic acid. This includes, for example, aldehyde oxidase genes derived from plants and preferably the gene of the present invention (an aldehyde oxidase gene described in items 1 to 7 above).
Transformation of a host cell by introducing such plasmid into said host cell can be effected by a method generally used in the field of genetic engineering.
When the host cell is a plant cell, it can be effected, for example, by a method generally used in the field of plant genetic engineering and the field of plant tissue cultivation as described above.
The transformation of a plant by introducing the gene of the present invention may bring about enhancement of generally known physiological action of auxin or supression of the same. For example, by enhancing the activity of auxin through a sense gene, elongation growth and differentiation to vascular bundle of the host cell can be accelerated resulting in growth acceleration of a plant and enhanced capacity of storing assimilation products. As a result, early maturing of crops, enlargement of harvest such as fruits and improvement in yield or quality can be expected and realized. To the contrary, by suppressing the activity of auxin through a sense gene, spindly growth of a plant is prevented and a plant capable of growing under improper environmental conditions such as insufficient insolation can be bred. Further, by adequately controlling growth, dwarfing of crops becomes possible and application, for example, to prevention of lodging of rice plants and shortening of cut flowers become possible. As a result, improvement in yield and quality can be expected.
Addition of hormone to the medium is generally essential for aseptic cultivation of cells or tissue of a plant. When auxin activity in a plant is enhanced by introducing and expressing the gene of the present invention thereby increasing production of aldehyde oxidase in a transformant, said plant is expected to be in a state in which capacity of cell proliferation, differentiation and individual regeneration in the sterile culture is enhanced. Therefore, it is possible to create a so-called easily cultured strain and this is useful in the production of nursery plant of virus-free crops for which tissue culture-nucleotide mass culture is conducted and garden crops such as flower and ornamental plants.